O-linked beta-N-acetylglucosamine (O-GlcNAc) is both an abundant and dynamic posttranslational modification similar to phosphorylation that occurs on serine and threonine residues of cytosolic and nuclear proteins in all metazoans and cell types examined, including cardiovascular tissue. Since the discovery of O-GlcNAc more than 20 years ago, the elucidation of O-GlcNAc as a posttranslational modification has been slow, albeit similar to the rate of acceptance of phosphorylation, because of the lack of tools available for its study. Identifying O-GlcNAc posttranslational modifications on proteins is a major challenge to proteomics. The recent development of mild beta-elimination followed by Michael addition with dithiothreitol has significantly improved the site mapping of both O-GlcNAc and O-phosphate in functional proteomics. beta-Elimination followed by Michael addition with dithiothreitol facilitates the study of the labile O-GlcNAc modification in the etiology of disease states. We discuss how recent technological innovations will expand our present understanding of O-GlcNAc and what the implications are for diabetes and cardiovascular complications.